Integrin-mediated cell adhesion permits proper interactions between key cytoskeletal and/or signaling proteins within the focal adhesion complex (FA) that are required for efficient signal transduction in the ERK/MAP kinase pathway. Many FA protein interactions have been identified, however the important molecular interactions required for adhesion regulation of signaling are still unclear. Recently novel approaches have emerged that will be useful in addressing this problem. This includes use of RNA interference (RNAi), as well as intracellular delivery of peptides or protein domains that block protein-protein interactions. These approaches will be utilized in a systematic manner to identify key protein-protein interactions required for efficient adhesion regulation of signaling. The FA proteins talin and paxillin have been selected for initial study based on their interactions with proteins that influence the cytoskeleton, cell motility and signaling. RNAi will be applied to ablate talin and paxillin. Synthesis and delivery of cell permeable peptides representing the sequences important for interactions between talin and beta1 integrin and those between paxillin and FAK or vinculin will be utilized to perturb those specific interactions. Expression and delivery of talin and paxillin domains will also be used to inhibit protein interactions. The application of these novel tools, in combination with confocal microscopy and cellular assays for adhesion, spreading, migration and signaling, will determine FA protein interactions important for integrin-regulated signaling to the ERK/MAP kinase cascade, and to cytoskeletal reorganization and cell motility.